Nearly all mitochondrial proteins are synthesized in the cytoplasm and then imported into the organelle. We propose to address some major unresolved mechanistic and structural issues about the import process, such as whether substrates are translocated across or inserted into the IM by aqueous pores, by positioning fluorescent or photoreactive probes at specific sites in matrix or membrane proteins (substrates). In this way we can directly monitor substrate environment and interactions during import. The probes will be incorporated into substrates at specific sites by in vitro translation using modified aminoacyl-tRNAs, an approach we originated and have used successfully to examine protein processing at the ER membrane. By creating import intermediates, we can position a probe at a specific location within the outer membrane, intermembrane space (IMS), inner membrane (IM), or matrix. This approach will allow us to determine whether imported proteins pass through the nonpolar interior of the IM or through a gated aqueous pore in the IM. Fluorescent probes in the substrate will be positioned within the IM and their fluorescence lifetimes will reveal whether they are in an aqueous or nonaqueous milieu. Collisional quenching experiments will determine if the substrate is exposed to the IMS, as well as when the substrate enters the matrix or IM. Photocrosslinking will be used to identify both the proteins that interact with a matrix or IM protein during its import, and also when each contact occurs. Fluorescence and photocrosslinking studies will reveal whether the transmembrane segments of an IM protein are inserted into the IM simultaneously or sequentially. The complexes formed between substrate proteins and tiny Tim proteins in the IMS will be characterized using both fluorescence and photocrosslinking, and the affinity of the tiny Tim proteins for Tim23p will be determined spectroscopically. Our approach to examining mitochondrial import will provide a unique perspective and novel insights into the mechanisms by which protein translocation and integration are accomplished at the mitochondrion without disrupting the integrity of the IM.